Biochemical and biological assays are designed to test for activity in a broad range of systems ranging from protein-protein interactions, enzyme catalysis, small molecule-protein binding, to cellular functions.
In general, fluorescent, chemiluminecent and other assay formats comprise three distinguishable response ranges. Where the amount of analyte being assayed is within the dynamic range of the assay, the reported signal will be dependent upon the amount of analyte present. Where the amount of analyte exceeds the dynamic range of the assay, saturation will occur and the reported signal will not be indicative of the true analyte concentration. Likewise, where the amount of analyte present in the sample falls below the threshold of the assay's dynamic range, the assay may be insufficiently sensitive to the actual analyte concentration, and the reported signal will also not be indicative of the true analyte concentration.
Two approaches have conventionally been employed to address this problem. In the first, multiple dilutions or concentrations of a sample are made and then assayed for a defined time period and the results are evaluated against that of a “standard curve” of assay results obtained with analyte of varying but known concentration. In the second approach, an amount of sample is assayed for multiple times, and results falling within the dynamic range of the assay are used to calculate the analyte's concentration (see, for example, U.S. Pat. No. 5,306,468 (Anderson et al.), U.S. Pat. No. 6,212,291 (Wang et al.)).
U.S. Pat. Nos. 6,270,695; 6,218,137; 6,139,782; 6,090,571 and 6,045,727 (Akhavan-Tafti, et al.) and U.S. Pat. Nos. 6,045,991; 5,965,736; 6,580,963 and 5,772,926 (Akhavan-Tafti) indicate the possibility of making multiple exposures in chemiluminescent assays. The use of multiple exposures in photography is also known (see, for example, U.S. Pat. No. 6,177,958 (Anderson) and U.S. Pat. No. 5,754,229 (Elabd)).
Microtiter or multi-well plates are becoming increasingly popular in various chemical and biological assays. High-density format plates, such as 384, 864 and 1536 well plates, are beginning to displace 96-well plates as the plate of choice. Many of the assays conducted in multiwell plates employ some type of light detection from the plate as the reporter for positive or negative assays results. Such assays include fluorescence assays, chemiluminescence assays (e.g., luciferase-based assays), phosphorescence assays, scintillation assays, and the like. In particular, with the advent of solid phase scintillating materials, capsules and beads, homogeneous scintillation proximity assays (SPA) are now being performed with increasing frequency in multiwell plates.
Detection of light signals from multiwell plates in the past has typically been done using plate readers, which generally employ a photodetector, an array of such photodetectors, photomultiplier tubes or a photodiode array to quantify the amount of light emitted from different wells. Such plate readers have been disclosed, for example, by (U.S. Pat. No. 4,810,096 (Russell, et al.) and (U.S. Pat. No. 5,198,670 (VanCauter, et al.)). Although plate readers can detect the total light from each well, they have a number of limitations. For example, plate readers are typically not capable of resolving discrete light sources in a single well, so they could not be used, for example, to differentiate light from different beads in one well. Further, most plate readers have fewer photodetectors than there are wells in the plate, so at least some wells must be read serially, adding to the time required to complete the assays. This becomes a substantial problem in assays where the light signal is so low that each well needs to be in the detection field for an extended period of time (e.g., tens of minutes). In addition, most currently-available plate readers have been designed for 96-well plates. Although some can be adapted for, e.g., 384-well plates, the adaptation does not result in any significant increase in throughput, since a 384-well plate going through a modified 96-well reader typically takes four times as long to read as a 96-well plate.
Another technique that has been applied to the detection of light from multiwell plates is imaging. Prior art imaging systems typically comprise a standard 50-55 mm fl.4 photographic lens coupled to a camera. While such systems can be used to image an entire multiwell plate, and theoretically provide resolution of discrete light points within individual wells, they have poor sensitivity, even when coupled to efficient cameras, so that many assays still require imaging times of tens of minutes or more. Other assays, such as SPA bead-based assays, cannot be performed at all due to lack of sensitivity. Further, images acquired with such systems suffer from vignetting and lateral distortion effects, making it difficult or impossible to compare signals from center portions of the plate with signals from lateral wells.
The demand for increased throughput during primary screening using less reagent is changing the way of drug discovery. High throughput screening in 96-well format plates is being replaced by the use of higher density plates, such as 384 and 1536-well formats. The analysis of radiometric assays by scintillation counters is becoming limiting since only 12 wells can be counted at a time. (Manzella S. M., et al. “A biphasic radiometric assay of glycogenin using the hydrophobic acceptor n-dodecyl-beta-D-maltoside,” Anal. Biochem. 1994 Feb. 1;216(2):383-91) (West B. C., et al. “Neutrophil uptake of vaccinia virus in vitro,” J. Infect. Dis. 1987 Octber ;156(4):597-606) (Boonkitticharoen V., et al. “Radiometric assay of bacterial growth: analysis of factors determining system performance and optimization of assay technique,” J. Nucl. Med. 1987 February;28(2):209-170).
Charge coupled devices (CCD) use a light-sensitive integrated circuit to store and display data for an image in such a way that each pixel (picture element) in the image is converted into an electrical charge, the intensity of which is related to a color in the color spectrum. Such devices have found use in chemical assays and radiologic imaging (see, for example, U.S. Pat. No. 5,306,468 (Anderson et al.), U.S. Pat. No. 6,212,291 (Wang et al.).
A CCD reads the light emitted through the electrode and the signal is sent to a microprocessor which converts the signal to the desired readout form. Data obtained (and, optionally, recorded) by the detection device is typically processed, e.g., by digitizing the image and storing and analyzing the image on a computer readable medium. A variety of commercially available peripheral equipment and software is available for digitizing, storing and analyzing a signal or image. For example, real-time binding and dissociation can be monitored visually or by video imaging, such as with a CCD camera and frame grabber software (U.S. Pat. No. 5,599,668 (Stimpson, et al.; U.S. Pat. No. 5,843,651 (Stimpson, et al.)).
CCD cameras are used for many applications in biochemistry and medicine (Dujardin F. H., et al., “Quantitative assessment of cortical bone remodelling from routine radiographs of total hip arthroplasty,” Med.Eng. Phys. 1996 September; 18(6): 489-94; Houze T. A., et al., “Detection of thymidylate synthase gene expression levels in formalin-fixed paraffin embedded tissue by semiquantitative, nonradioactive reverse transcriptase polymerase chain reaction,” Tumour Biol. 1997;18(1):53-68); Innocenti B., et al., “Imaging extracellular waves of glutamate during calcium signaling in cultured astrocytes,” J. Neurosci. 2000 Mar. 1;20(5):1800-8; Katanec D, et al., “Computer assisted densitometric image analysis (CADIA) of bone density in periradicular bone defects healing,” Coll Antropol—1998 December; 22 Suppl: 7-13; Nilsson H, et al., “Laser-induced fluorescence studies of the biodistribution of carotenoporphyrins in mice,” Br. J. Cancer 1997;76(3):355-64; O'Rourke B, et al., “High-speed digital imaging of cytosolic Ca2+ and contraction in single cardiomyocytes,” Am J Physiol 1990 July;259(1 Pt 2):H230-42); Peng Q., et al., “Correlation of distribution of sulphonated aluminium phthalocyanines with their photodynamic effect in tumour and skin of mice bearing CaD2 mammary carcinoma,” Br. J. Cancer 1995 September;72(3):565-74; Pope A. J., et al., “The detection of phthalocyanine fluorescence in normal rat bladder wall using sensitive digital imaging microscopy,” Br. J. Cancer 1999 November;64(5):875-9; Yasui T, et al., “Imaging of Lactobacillus brevis single cells and microcolonies without a microscope by an ultrasensitive chemiluminescent enzyme immunoassay with a photon-counting television camera,” Appl. Environ. Microbiol. 1997 November;63(11):4528-33; Zhang J. H., et al. “Development of a carbon dioxide-capture assay in microtiter plate for aspartyl-beta-hydroxylase,” Anal. Biochem. 1999 Jul. 1;271(2):137-42).
Although methods involving multiple exposure times have been used to identify the optimum desired exposure time in enzymatic assays, a need remains for automatable methods that can be used to process simultaneously not only multiple samples, but also multiple signal generation foci within each sample, each potentially reporting differing signals in the assay. The present invention addresses this need.